In a typical AlphaScreen assay, one biomolecule of interested is biotinylated and associates with the Streptavidin-coated Donor beads. The other biomolecule is c-myc-tagged, and associates with the AlphaScreen anti-c-myc Acceptor beads. If the two biomolecules bind to each other, the Donor and Acceptor beads are brought into proximity. Excitation of the Donor beads causes the release of singlet oxygen, which diffuses and triggers the emission of light from the Acceptor beads when in proximity. The amount of light is directly proportional to the degree of interaction. Competition formats are also possible, using the biotinylated c-myc probe provided in the kit.

Guide

Alpha has been used to study a wide variety of interactions, including protein:protein, protein:peptide, protein:DNA, protein:RNA, protein:carbohydrate, protein:small molecule, receptor:ligand, and nuclear receptor:ligand interactions. Both cell-based and biochemical interactions have been monitored, and applications such as phage display, ELISA, and EMSA (electrophoretic mobility shift assay) have been adapted to Alpha.

AlphaScreen® and AlphaLISA® are bead-based assay technologies used to study biomolecular interactions in a microplate format. The acronym “Alpha” stands for Amplified Luminescent Proximity Homogeneous Assay. The assay does not require any washing steps. Binding of proteins or other binding partners captured on the beads leads to an energy transfer from one bead to the other, ultimately producing a luminescent signal.